Immunogens for treatment of neoplastic and infectious disease

ABSTRACT

The present invention relates to prophylactic and therapeutic methods of immunization against neoplastic and infectious diseases. The invention provides a method for identification of novel immunogens and compositions of such immunogens that are useful for eliciting immune responses against antigens associated with neoplastic or infectious diseases.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/379,642, filed Mar.3, 2003,now U.S. Pat. No. 8,088,569 issued Jan. 3, 2012, and claimspriority to U.S. Ser. No. 60/360,720, filed Mar. 1, 2002, the contentsof which applications are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to prophylactic and therapeutic methods ofimmunization against neoplastic and infectious diseases. Morespecifically, the invention provides a method for identification ofnovel immunogens and compositions of such immunogens that are useful foreliciting immune responses against antigens associated with neoplasticor infectious diseases.

BACKGROUND OF THE INVENTION

Therapy for neoplastic disease has largely involved the use ofradiation, surgery and chemotherapeutic agents. However, thesetreatments, while beneficial in some cases, have marginal or no effectin many others. Furthermore, these approaches often have unacceptabletoxicity. In cases where the cancer has become metastatic, there isoften no effective curative treatment.

Specificity is a major problem with most anticancer agents. Ideally,anticancer agents would discriminate between host cells that arecancerous and host cells that are not cancerous. However, mostanticancer drugs are indiscriminate at this level and generally targetactively multiplying cells. Among the consequences are immunosuppressionand disruption of hematopoiesis, epithelial tissues (e.g., intestinalmucosa) and the reproductive system.

Heat shock proteins (Hsps) were originally observed to be expressed inincreased amounts in mammalian cells which were suddenly exposed tosudden elevations in temperature. Further experiments demonstrated thatsuch proteins are produced in response to various types of stress,including, for example, glucose deprivation. The family of heat shockproteins further includes homologous proteins that are constitutivelyexpressed. For example, whereas Hsp70 is not expressed at normaltemperatures, a constitutively expressed heat shock protein cognate(Hsc70) participates in protein translocation across membranes and otherfunctions. Most heat shock proteins are referred to as “molecularchaperones” in that they bind and stabilize proteins in their non-nativestates. For example, Hsps bind to nascent peptides emerging fromribosomes or extruded into the endoplasmic reticulum, at intermediatestages of folding, and assembly.

It has been observed that Hsps prepared from tumors in experimentalanimals are able to induce immune responses in a tumor specific manner.Evidence suggests that immunity is not conferred by the Hsp per se, butby peptides complexed with the Hsp.

Although autologous tumor-derived Hsp-peptide complexes are capable ofeliciting an immune response against cells of the same tumor, there aredrawbacks to use of such autologous complexes in vaccines for cancertreatment. For example, each patient would require a personalized orcustom vaccine. To prepare such a vaccine, it would be necessary toprovide Hsp preparations from a patients own tumor. Furthermore,ensuring availability of sufficient lisp preparations would necessitateculture of the tumor tissue are required and Hsp preparations from suchtumors are low. Although peptides might be synthesized and complexedwith Hsps in vitro, attempts to identify peptides of interest complexedwith Hsps have generally been unsuccessful. Consequently, significantclinical constraints are imposed with regard to the manufacture ofHsp-peptide preparations.

SUMMARY OF THE INVENTION

A novel method is provided for identifying immunogens that can be usedto treat or prevent a neoplastic or infectious disease. The method isparticularly advantageous for identifying useful immunogens that can beused to elicit immune responses against target antigens associated withneoplastic or infectious diseases that are not immunogenic and thatwould otherwise escape immune surveillance. Immunogens identified by themethod are administered to a patient in need of treatment for aneoplastic or infectious disease.

Accordingly, the invention provides a method of eliciting an immuneresponse against a target cell in a host by isolating first bindingsubstances that specifically bind to heat shock protein-peptidecomplexes differentially expressed by the target cell relative to asecond cell, isolating second binding substances that specifically bindto the first binding substances, and immunizing the host with atherapeutically effective amount of the second binding substances. Invarious embodiments of the invention, the first and second bindingsubstances can be mixtures of binding substances of a single species. Inone embodiment, the binding substances can be obtained by screeninglibraries of phage displaying binding proteins such as antibodyfragments or peptides. In another embodiment, the binding substances canbe obtained by screening libraries of synthetic compounds.

The invention provides methods of obtaining binding substances thatmimic peptides that are complexed with heat shock proteins in targetcells. The peptide mimics (“mimotopes”) are used as to immunize the hostagainst the target cells. Target cells include, for example, neoplasticcells and infected cells.

The invention provides vaccines that incorporate the mimotopes. Themimotopes are incorporated by routine methods, such as covalentconjugation to earners and non-covalent binding to heat shock proteins.

The basis of the invention is a method for rapid identification ofpeptides or other molecules that contain mimotopes of target antigensdifferentially present on diseased cells. Advantageously, there is norequirement that any target antigen associated with the diseased cell bespecifically known or identified.

According to the method, a first population of binding substances isscreened to enrich or separate a subpopulation that binds to heat shockprotein-peptide complexes obtained from a diseased cell or tissue and isrelatively unreactive with heat shock protein-peptide complexes obtainedfrom normal tissue. The first subpopulation can consist of multiplehsp-peptide complex-specific binding substances, or just one.

A second population of binding substances is then screened against thefirst subpopulation of binding substances to enrich or identify membersof the second population that bind to the first subpopulation. Thesecond subpopulation includes substances that both mimic epitopespresent in the diseased cell or tissue and are immunologically active.

Thus, the second subpopulation of binding substances identified by themethod comprises determinants which are useful for eliciting immuneresponses against antigens associated with the diseased cell or tissue.The antigens can be associated with neoplastic diseases or infectiousagents. The peptides are particularly useful for eliciting immuneresponses against antigens which are otherwise unknown, poorlyimmunogenic, or to which a host is tolerized or becomes tolerized as adisease progresses.

Antigens which are poorly immunogenic include self antigens. An immuneresponse directed against a self antigen can be particularlyadvantageous where the self antigen is displayed primarily or solely ontumor cells. Non-self antigens may also be poorly immunogenic wherethere is a high degree of similarity between the non-self antigen and aself antigen. The response against the non-self antigen may be inhibitedby self-tolerance mechanisms. For example, the success of humanpapilloma virus (HPV) infection appears due in part to avoidance of thehost's immune surveillance system that would otherwise respond to theforeign viral oncoproteins and stem the spread of HPV infection. Theimmune system avoidance is thought to be related to amino acid sequencesimilarity between HPV proteins and host proteins when evaluated on thesize scale of peptides that can be presented by Class I or Class II MHC.In such cases, a method for eliciting immunity that avoidsself-tolerance is useful.

Mimotope containing peptides identified by the invention areadministered to subjects by methods that are well known in the art. Inaddition, the peptides can be administered by the usual routes instandard pharmaceutical compositions. The peptides can also beadministered in mixtures and covalent or non-covalent complexes withheat shock proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a panning strategy for screening single chain antibodies(scFvs) that bind preferentially to gp96-peptide complexes from Mat-LyLucells.

FIG. 2 shows a series of scFv-phage clones that are bind preferentiallyto gp96-peptide complexes from MAT-LyLu tumors from Copenhagen rats ascompared to complexes isolated from tumor free tissue from the sameanimal.

FIG. 3 shows the reactivity of five scFv antibodies with gp96-peptidecomplexes from MAT-LyLu cells as compared to normal tissue (liver,prostate) and a parental non-metastatic tumor line (Dunning G).

FIG. 4 shows a 170 kD protein precipitated from a MAT-LyLu cell lysateby scFv E6 (Lane 2) as compared to whole lysate (Lane 1) and scFv E6alone (Lane 3).

FIG. 5 shows a panning strategy for screening peptide mimics of theantigen that binds to scFv E6.

FIG. 6 shows reactivity with scFv E6 of peptide-phage isolated from alibrary displaying 15-mer peptides. B11 is a non-specific control scFv.

FIG. 7 shows the reactivity with scFv E6 of peptide-phage isolated froma library displaying 12-mer peptides containing a disulfide bridge. B11is a non-specific control scFv.

FIG. 8 shows immune reactivity of antisera from Copenhagen ratsimmunized with peptide LX-8 or X15-7. There is no apparentcross-reactivity with LX-8 of antisera from rats immunized with X15-7,or vice versa.

FIG. 9 shows binding inhibition by peptides LX-8 and X15-7 of binding ofanti-E6-X15-7 to X15-7 (FIG. 9 a) and anti-E6-LX-8 to LX-8 (FIG. 9 b).

FIG. 10 shows the effect of vaccination of synthetic peptides LX-8b orX15-7 on tumor progression.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to substances and methods used to identifysubstances that are useful for inducing host immune responses againsttarget antigens of interest, particularly cancer antigens and antigensfrom infectious agents that would be beneficial targets of a vigorousimmune response, but that are poorly immunogenic. To overcome poorimmunogenicity, the invention provides substances identified fromlibraries of epitope mimics that elicit active immune responses againstthe target antigen.

In an embodiment of the invention, candidate target antigens arepeptides complexed with Hsps. As used herein, the term “heat shockprotein” is used to encompass both proteins that are induced in responseto stress conditions and homologs of such proteins that areconstitutively expressed. Heat shock proteins of the invention include,but are not limited to gp96 (grp94), BiP (grp78), hsp70, hsc70, hsp60,hsp40, hsp90. In preferred embodiments, the heat shock protein is human.

The method is applicable to any cells or antigens of interest. Where animmune response against a target cell is desired, it is sufficient toselect a target cell without further consideration for a particularexpressed antigen. This is because the method allows for identificationof mimotopes for any antigen that is differentially expressed by thetarget cell relative to a normal cell or control cell. Where mimotopesto a particular antigen are desired, a cell that expressed that antigencan be used. For example, a target cell can be engineered that expressesa target antigen of interest by transfecting and expressing a controlcell line with a nucleic acid that encodes the target antigen ofinterest. Except for the target antigen of interest and any otherproteins that my be expressed as a result of the presence of the targetantigen in the cell, the target cell will express substantially the sameproteins as the untransfected control cell.

The method is particularly useful for targeting diseased cells (e.g.,neoplastic cells, infected cells) that express antigens which areexpressed in lower amounts or not at all in other tissue. Examples oftarget cells include cells from a neoplastic disease, including but notlimited to a sarcoma, a lymphoma, a leukemia, or a carcinoma, and inparticular, with melanoma, carcinoma of the breast, carcinoma of theprostate, ovarian carcinoma, carcinoma of the cervix, colon carcinoma,carcinoma of the lung, glioblastoma, astrocytoma, etc. Alternatively,the target cell can be infected by, for example, a virus, a mycoplasma,a parasite, a protozoan, a prion and the like.

Target antigens of particular interest are those associated with,derived from, or predicted to be associated with a diseased cell such asa neoplastic or infected cell. Accordingly, target antigens can be from,for example, a human papilloma virus (see below), a herpes virus such asherpes simplex or herpes zoster, a retrovirus such as humanimmunodeficiency virus 1 or 2, a hepatitis virus, an influenza virus, arhinovirus, respiratory syncytial virus, cytomegalovirus, adenovirus,Mycoplasma pneumoniae, a bacterium of the genus Salmonella,Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia,Klebsiella, Vibrio, Mycobacterium, amoeba, a malarial parasite,Trypanosoma cruzi, etc. In addition to tumor antigens and antigens ofinfectious agents, mutations of tumor suppressor gene products such asp53, or oncogene products such as Ras may also provide target antigensto be used according to the invention. The target antigen can be a selfantigen, for example one associated with a cancer or neoplastic disease.

Generally, target antigens of the invention are present in diseasedcells or tissue, but not in normal cells or tissue. However, it will berecognized that certain target antigens may be expressed in normaltissue in, for example a developmental or gender specific manner. Forexample, a host may be tolerized to a self-antigen that normally isexpressed at an early stage of development. When that antigen isaberrantly expressed at another point in time, it can be an ideal targetantigen. In another example, a target antigen of interest may be presentin normal tissue of one sex, but not the other. This method furtherapplies where a target antigen is expressed in diseased tissue, butnormal tissue of the same cell type has been removed from the host. Forexample, following prostatectomy, any prostate specific protein is apotential target antigen for eliciting an immune response directedagainst metastatic cells from a prostate cancer, because normal hosttissue that would be targeted by the immune response has already beenremoved. The invention is also applicable where a target antigen isexpressed in relatively large amounts in diseased cells, and smallamounts in normal cells. Here, an the intent is to elicit an immuneresponse that is effective against cells expressing the target antigenin large amounts (e.g., having a dense population of a cell surfaceantigen), but that has little or no effect on cells expressing thetarget antigen in small amounts.

In an embodiment of the invention, binding substances are identifiedthat are specific for hsp-peptide complexes from diseased cells ortissue. Binding substances include any synthetic and naturally occurringmolecules that are specific for hsp-antigen complexes from the diseasedcells or tissue. Examples include proteins, fragments thereof,polypeptides, polynucleotides, and synthetic analogs. Because thebinding substances are simply a source of “binding sites,” there is norequirement that the sequences be derived from a natural source, or thatthe structures resemble any naturally occurring protein. Syntheticanalogs include, for existence, polypeptide- and polynucleotide-likepolymers having modified backbones, side chains, and/or bases. In apreferred embodiment, the binding substances are antibodies andantibody-like molecules. Antibodies include complete immunoglobulins andfragments thereof that retain specific binding characteristics. Antibodyfragments include, e.g., Fabs, Fvs, and scFvs. Antibody-like moleculesinclude, e.g., molecules comprising Ig-like domains such as T cellreceptors and the like. The antibodies can be classical monoclonalantibodies developed by cell fusion techniques, and screened fordifferential binding to disease-associated hsp-peptide complexes.Alternatively the antibodies can be populations of polyclonal antibodiesraised against hsp-peptide complexes from diseased cells. Prior to use,such populations would be pre-bound to hsp-peptide complexes from normaltissue to remove components that are not disease specific.

In a preferred embodiment, synthetic antibody libraries are employed. Aparticularly preferred method for making and screening syntheticlibraries is phage display, which conveniently allows for positive andnegative screening steps and amplification of elements between screeningsteps. It is noted that any sort of binding substance can be used

Antibody libraries are generally screened by a method that enriches forelements that bind to disease specific targets, and removes elementsthat bind to targets present in normal tissue. It is characteristic ofphage display technology that enrichment is enhanced by repetition ofscreening steps. Thus, in certain embodiments of the invention, multiplerounds of positive and negative screens are performed. The number ofrounds depends on the desired binding substances. Where a single bindingsubstance is desired, the screening process is essentially complete whenthat substance is identified. Where a diverse population of bindingproteins is desired, for example, where multiple target antigens exist,it is beneficial to repeat screening steps until a substantialproportion of the binding proteins are disease specific. Generally, atleast half of the binding proteins will be disease specific. Preferably,at least 80% of the binding proteins will be disease specific. Morepreferably, at least 90% will be disease specific.

Immunogenic substances of the invention are identified by their abilityto bind specifically to the above described binding substances.Immunogenic substances are generally identified by generating librariesand screening for desired binding characteristics. Preferred immunogenicsubstances of the invention are peptides, but the methods and examplesdisclosed are generally applicable to any epitope library. In anembodiment of the invention, peptides to be screened are encoded byfilamentous phage and expressed as part of a phage coat protein. Thetechnique of phage display is well known in the art, and allows foramplification of the library in between rounds of screening.

Peptides screened and identified by the method can be relatively short,and are generally 30 amino acids or less. Immunogenic substances arelarge enough to comprise a single T cell or B cell epitope. Peptideimmunogens are preferable from 4 to 20 amino acids, and more preferablyfrom 7 to 15 amino acids. The peptides can have flexible or constrainedsecondary structures. The flexibility of linear peptides is constrained,for example, by incorporation of proline or by disulfide bonds formedbetween cysteine residues.

Other types of libraries may also be employed, for example, any sort ofsynthetic polymers that can be screened and optionally, their structureor sequence determined. Numerous methods have been devised forgeneration of chemical diversity and mass screening of libraries. Tosimplify identification of library elements of interest, many librarieshave the feature that synthesis steps are encoded. For example, in phagedisplay, the displayed peptide is encoded in the genome encapsulated bythe phage particle. Amino acid sequences of peptides identified byscreening procedures are easily determined by sequencing a smallpredetermined part of the genome.

In another example, it has been demonstrated that peptides can begenerated in numbers several orders of magnitude greater than byconventional one-at-a-time methods by synthesis on polyethylene rods orpins, arranged, for example, in a microtiter plate format. The pintechnology is representative of techniques that generate libraries ofsingle compounds in a spatially-differentiated manner. An alternativeapproach, to rapidly prepare large mixtures of compounds, is thesplit-pool approach (e.g., Houghten, R. A., 1985, Proc. Natl. Acad. Sci.U.S.A. 82:5131-5135) where a solid support material (e.g., beads) isphysically segregated into equal portions for coupling to each of theindividual initial reactants. This affords uniform coupling sincecompetition between reactants is eliminated. The individual polymers arecombined in a single vessel for washing and deprotection and thendivided again into individual portions for, the next coupling. Usingthis approach, a complete set of possible molecular combinations israpidly prepared in approximately equimolar amounts. Coincident withcoupling reactions, “identifier” tags can be attached to the solidsupport material. The structure of the molecule on any bead identifiedthrough screening is obtained by decoding the identifier tags. Numerousmethods of tagging the beads have now been reported.

Alternatives to peptide libraries include production of polymers ofpeptide-like and small organic molecules. For example, peptide librariesare a collection of N-substituted glycines as peptide monomers which areassembled in a modular fashion. (Zuckermann, R. N. et al, 1994, J. Med.Chem. 37:2678-2685.) The structures of the resulting compounds areunique, display unique binding properties, and incorporate the importantfunctionalities of peptides in a novel backbone. Furthermore, studiessuggest this class of compounds is resistant to enzymatic breakdown.

Thus, the invention can be practiced in a variety of ways. In a firstnon-limiting example of the invention, a single binding protein isidentified that differentially binds to a hsp-peptide complex from adiseased cell. The binding protein is used to identifymimotope-containing peptide immunogens, and one of those peptideimmunogens is selected for use in a treatment regimen. In a secondexample, a single binding protein is identified, but of themimotope-containing peptide immunogens that are recovered, all or amultiplicity or the peptides are selected for treatment. In a thirdexample, multiple binding proteins are identified and then used toobtain mimotope-containing peptides. It may be advantageous to furthercharacterize the binding proteins and mimotope-containing peptides(e.g., to obtain amino acid sequence information), but furthercharacterization is not critical to practice of the invention.

It is expected that individual mimotope-containing peptides will exhibitdifferent degrees of immunogenicity. Immunogenicity can depend, forexample, on the degree of sequence similarity between the peptide andthe host proteome. Sequence similarity is easily determined by methodsthat are well known in the art. Immunogenicity can also depend on thegenetic makeup of the host. For example, a variety of MHC alleles arepresent in the human population, and differences in peptide presentationto the immune system are observed depending on the MHC allele and thesequence of the peptide. Such differences in peptide presentation affectboth the strength and the type of immune response (e.g., humoral, cellmediated, cytolytic). Databases and analysis methods have been developedthat disclose sequence preferences of Class I and Class II MHC allelesand are well known to those of skill in the art. See, e.g., Rammensee etal., Immunogenetics 41:178-223 (1995).

In a preferred embodiment, peptide immunogens of the invention areconjugated to immunogenic carriers. As used herein, “immunogeniccarrier” means a molecule which can assist T lymphocyte activation andhelper T cell participation. Hence, to stimulate an immune responseagainst an a peptide immunogen, the peptide can be covalently linked(i.e., conjugated) to such an immunogenic carrier. An immunogeniccarrier of the invention can be a protein or portion of a protein whichstimulates or enhances the immune response of the subject. Examples ofimmunogenic carrier proteins are KLH and BSA. Immunogenic carriers alsoinclude polypeptides that are promiscuous Class II activators (see,e.g., Panina-Bordignon et al., 1989). Conjugate linkages are made bymethods well known to those of skill in the art.

In addition to immunogenic proteins and polypeptides which comprise a Tcell epitope, carriers can also be constructs to which other immunogenicmoieties (e.g., cytokines, polypeptides bearing T cell epitopes, etc.)can be linked. Branched constructs such as lipo-thioester and branchedpolylysine allow for multiple covalent linkages of such immunogenicmoieties as well as conjugation of multiple polysialic acid polymers.Carriers further include proteins and polypeptides which have beenmodified by the covalent addition of immunogenically active moieties.Alternatively, the peptide immunogens can be incorporated into longersequences of amino acids. The additional sequences can, for example,confer a desired function, such as the ability to bind to a heat shockprotein. In certain embodiments, tandem arrays will be produced thatcomprise multiple copies of one peptide immunogen or comprise multiplepeptide immunogens.

Immunogen-carrier conjugates are typically formulated with theadjuvants, which generally promote nonspecific immune responses. Whilecommercially available pharmaceutically acceptable adjuvants arelimited, representative examples of adjuvants include BacilleCalmette-Guerin, BCG, or the synthetic adjuvant, QS-21 comprising ahomogeneous saponin purified from the bark of Quillaja saponaria,Corynebacterium parvun, (McCune et al., 1979, Cancer 43:1619), andIL-12.

Immunogenic compositions comprising immunogens identified according tothe invention may be administered to a subject using either a protein ornucleic acid vaccine so as to produce in the subject, an amount of theselected immunogen which is effective in inducing a therapeutic immuneresponse against the target antigen in the subject. The subject may be ahuman or nonhuman subject. The term “therapeutic immune response”, asused herein, refers to an increase in humoral and/or cellular immunity,as measured by standard techniques, which is directed toward the targetantigen. Preferably, the induced level of immunity directed toward thetarget antigen is at least four times, and preferably at least 16 timesthe level prior to the administration of the immunogen. The immuneresponse may also be measured qualitatively, wherein by means of asuitable in vitro or in vivo assay, an arrest in progression or aremission of a neoplastic or infectious disease in the subject isconsidered to indicate the induction of a therapeutic immune response.

In the methods of the present invention, a therapeutically effectiveamount of an immunogen (e.g., a second binding substance) isadministered to a mammal in need thereof. The term “administering” asused herein means delivering the antibodies of the present invention toa mammal by any method that may achieve the result sought. They may beadministered, for example, intravenously or intramuscularly. The term“mammal” as used herein is intended to include, but is not limited to,humans, laboratory animals, domestic pets and farm animals.“Therapeutically effective amount” means an amount of antibody of thepresent invention that, when administered to a mammal, is effective inproducing the desired therapeutic effect, such as inhibiting kinaseactivity.

Compositions comprising antigenic peptides of the invention may beadministered cutaneously, subcutaneously, intravenously,intramuscularly, parenterally, intrapulmonarily, intravaginally,intrarectally, nasally or topically. The composition may be delivered byinjection, orally, by aerosol, or particle bombardment.

Compositions for administration may further include various additionalmaterials, such as a pharmaceutically acceptable carrier. Suitablecarriers include any of the standard pharmaceutically accepted carriers,such as phosphate buffered saline solution, water, emulsions such as anoil/water emulsion or a triglyceride emulsion, various types of wettingagents, tablets, coated tablets and capsules. Typically such carrierscontain excipients such as starch, milk, sugar, certain types of clay,gelatin, stearic acid, talc, vegetable fats or oils, gums, glycols, orother known excipients. Such carriers may also include flavor and coloradditives or other ingredients. The composition of the invention mayalso include suitable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions may be in thefoam of liquid or lyophilized or otherwise dried formulations and mayinclude diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength, additives such as albumin or gelatinto prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80,Pluronic F68, bile acid salts), solubilizing agents (e.g. glycerol,polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol,parabens), bulking substances or tonicity modifiers (e.g., lactose,mannitol), covalent attachment of polymers such as polyethylene glycolto the protein, complexing with metal ions, or incorporation of thematerial into or onto particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, hydrogels, etc. or ontoliposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts, or spheroplasts. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance.

As an alternative to direct administration of a peptide immunogen, oneor more polynucleotide constructs may be administered which encode thepeptide immunogen in expressible form. The expressible polynucleotideconstructs are introduced into cells in the subject using ex vivo or invivo methods. Suitable methods include injection directly into tissueand tumors, transfecting using liposomes, receptor-mediated endocytosis,particle bombardment-mediated gene transfer, and other methods of genetransfer. The polynucleotide vaccine may also be introduced intosuitable cells in vitro which are then introduced into the subject. Toconstruct an expressible polynucleotide, a region encoding the peptideantigen is prepared and inserted into a mammalian expression vectoroperatively linked to a suitable promoter such as the SV40 promoter, thecytomegalovirus (CMV) promoter, or the Rous sarcoma virus (RSV)promoter. The resulting construct may then be used as a vaccine forgenetic immunization. The nucleic acid polymer(s) could also be clonedinto a viral vector. Suitable vectors include but are not limited toretroviral vectors, adenovirus vectors, vaccinia virus vectors, poxvirus vectors and adenovirus-associated vectors. Specific vectors whichare suitable for use in the present invention are pcDNA3 (In-Vitrogen),plasmid AH5 (which contains the SV40 origin and the adenovirus majorlate promoter), pRC/CMV (InVitrogen), pCMU II (Paabo et al., EMBO J.5:1921-1927 (1986)), pZip-Neo SV (Cepko et al., Cell 37:1053-1062(1984)) and pSRα (DNAX, Palo Alto, Calif.).

Throughout this application, various publications, patents, and patentapplications have been referred to. The teachings and disclosures ofthese publications, patents, and patent applications in their entiretiesare hereby incorporated by reference into this application to more fullydescribe the state of the art to which the present invention pertains.

It is to be understood and expected that variations in the principles ofinvention herein disclosed may be made by one skilled in the art and itis intended that such modifications are to be included within the scopeof the present invention.

The examples which follow further illustrate the invention, but shouldnot be construed to limit the scope in any way.

EXAMPLES Example 1 Identification of Prostate Cancer-Specific Antigenand Induction of Immunity

In this example, a prostate cancer specific antigen is first identifiedby developing a scFv that differentially binds to gp96-antigen complexesfrom tumor cells and isolating and characterizing the complete proteinthat comprises the epitope to which the scFv binds. Mimotopes of theprostate specific antigen are obtained by screening for peptides thatbind to the same scFv.

With respect to prostate cancer, several cell lines exist having variedbiological characteristics with respect to tumorigenic potential,hormone sensitivity and metastatic properties. Dunning G is an androgensensitive tumor line which is slow growing and possesses no metastaticability. Dunning H is a heterogenous population of cells, passaged astumors in castrated rats. Mat-LyLu is a subline of Dunning H which isandrogen insensitive, has a short doubling time, and metastasises to thelung and lymph nodes.

A binding protein is developed that is specific for a heat shockprotein-peptide complex from Mat-LyLu, but which does not bind to a heatshock protein-peptide complex from normal tissue. First, a library ofsingle chain Fv (scFv) antibodies displayed on phage is panned againstgp96-peptide complexes from liver. The library comprises heavy chainsencoded by 49 human V_(H) genes with synthetic D and J_(H) sequences.Thus, unlike V_(H)-CDR1 and V_(H)-CDR2, which are encoded in the V_(H)genes, V_(H)-CDR3 is synthetic. As identical light chain is used in allmembers of the scFv library. Unadsorbed phage are recovered and thepanning process is repeated two more times to remove phage that bind tocomplexes from normal tissue. Unadsorbed phage are then panned overtumor derived heat shock protein-peptide complexes. Adsorbed phage arerecovered and amplified.

Three rounds of differential panning are then performed. In each round,amplified phage are first panned over liver derived gp96-peptidecomplexes. Unadsorbed phage are then panned over tumor-derivedgp96-peptide complexes. Adsorbed phage are recovered and amplified.

Adsorbed phage at the last stage of panning are eluted and individualphage clones are tested for reactivity to gp96-peptide complexes byELISA. Reactivity against both tumor-derived gp96-peptide complexes andnormal liver-derived gp96-peptide complexes is determined. The phagerecovered in the last step display a significant binding differential.In most cases, binding of the phage clones to liver-derived complexes isnot above background.

Phage clones are then tested to determine whether the gp96-peptidecomplexes to which they bind are tumor specific. ELISA assays areperformed using gp96-peptide complexes purified from Mat-LyLu, DunningG, liver and normal prostate cells. The phage clones are specific for agp96-peptide complex made by Mat-LyLu cells that is not present innormal cells or in Dunning G.

The heavy chain sequences of two scFvs that bind to tumor derived gp96complexes are determined. Both have V_(H)3 heavy chains and the sameV_(H)-CDR3 sequence (GKYIRSV: SEQ ID NO:4). One of the scFvs (E6) istested and found to precipitate a 170 kD protein from MAT-LyLu cells.The protein is analyzed for amino acid composition, sequence andcleavage patterns and identified as heavy polypeptide 9 of non-musclemyosin (NCBI Accession #6981236).

Two phage display libraries are panned to identify peptides that bind toMAT-LyLu specific scFv E6. One library comprises 12-mer peptide inserts(the “LX-8 library”), including two cysteine residues and a sulfidebridge. The second library comprises “linear” 15-mer peptide inserts(the “X-15 library”).

scFv E6 is biotinylated and bound to streptaviclin coated 96 wellmicrotiter plates for 1 hr. at 4° C. The plates are washed and blockedwith 300 μl blotto (5% milk, 10 mM EDTA) containing 0.12 mM biotin atroom temperature for 2 hr. To each well is added 50 μl blotto and 100 μlTBS containing 10¹² virions. The plates are incubated at for 4 hr. at 4°C. The wells are washed with TBS and bound phage are eluted by adding 35μl of elution buffer (0.1 M HCl, pH 2.2) and incubating at roomtemperature for 10 min. Eluted phage are neutralized with 6.6 μl of 1 MTris, pH 9.1 and amplified using E. coli K91. Panning and amplificationsteps are repeated until four rounds of panning have been performed.

Reactivity of several peptide phage from the X-15 library and the LX-8library to the scFv is determined by ELISA. Peptides are identified ineach library that bind to scFv E6 but not to a control antibody. Theamino acid sequence of a peptide from the LX-8 library is YCQEGDAPRLCL(“BTE6-LX-8b”; SEQ ID NO:1). The sequences of two peptides from the X-15library are determined to be YQPPSDALRWILRLQ (“BTE6-X15-4”; SEQ ID NO:2)and GQWQSGDRYWMETST (“BTE6-X15-7”; SEQ ID NO:3).

A peptide from each of the libraries is synthesized according to theabove sequences and conjugated to KLH using 0.25% glutaraldehyde.Copenhagen rats are immunized sub-cutaneously (day 0) with eitherpeptide (100 μg peptide/animal) in complete Freund's adjuvant (CFA) or40 μg MLL-derived gp96 in PBS. On day 14, the rats are boosted with 50μg peptide/animal in incomplete Freund's adjuvant (IFA) or 40 μgMILL-derived gp96 and challenged subcutaneously with 10,000 MAT-LyLucells/animal. A second boost (50 μg/animal in WA) is administered on day21.

On day 21, prior to boost, and again on day 28, immunized rats are bledand screened for antibody production by ELISA. Immunization with each ofthe peptides elicits an immune response that is peptide specific. Forexample, while BTE6-LX-8b antisera reacts with BTE6-LX-8b peptide, thereis no cross-reactivity with BTE6-X15-7 peptide. In contrast,immunization with MLL-derived gp96 does not elicit a measurable immuneresponse.

Two weeks after subcutaneous challenge (day 28), tumors arising fromMAT-LyLu cells are evaluated for size. Large masses are found to developin control rats immunized with MILL-derived gp96. In contrast, there islittle, if any, tumor progression in rats immunized with BTE6-LX-8b orBTE6-X15-7.

Example 2 Induction of Immunity Against Multiple and UnspecifiedAntigens

In this example, mimotopes of mouse mammary tumor specific antigens aresought. No attempt is made to determine the identity of any cancerspecific antigen.

A population of binding proteins is developed that binds to hsp-peptidecomplexes from mammary tumor cells, but not to hsp-peptide complexesfrom normal mammary tissue. A library of single chain Fv antibodiesdisplayed on phage is panned multiple times against gp96-peptidecomplexes from diseased and normal tissue according to the protocol ofExample 1. Members of the recovered population are tested for bindingspecificity. Over 90% of the recovered scFv-phage bind specifically forhsp-peptide complexes from breast tumor cells.

As in Example 1, two peptide-phage display libraries are panned toidentify peptides that bind to the recovered, amplified population ofscFv-phage.

The sequences of ten peptides from each of the libraries are determined,and The peptides are synthesized and conjugated to KLH using 0.25%glutaraldehyde. Mice are immunized sub-cutaneously (day 0) with amixture of the 20 conjugates (50 μg peptide/animal) in complete Freund'sadjuvant (CFA) or a conjugate of a non-specific peptide (control). Onday 14, the mice are boosted with 50 μg peptide/animal in incompleteFreund's adjuvant (IFA) and challenged subcutaneously with 10,000 tumorcells/animal. A second boost (50 μg/animal in IFA) is administered onday 21.

Two weeks after subcutaneous challenge (day 28), tumors arising frommammary tumor cells are evaluated for size. Cell masses are found todevelop in control rats immunized. In contrast, there is little, if any,tumor progression in rats immunized with the mixture of 20 conjugates.

What is claimed is:
 1. A method of making an immunogenic compositionagainst a target cell or an antigen thereof, wherein the target cell isa tumor cell or an infected cell, comprising: a) isolating first bindingsubstances that specifically bind to heat shock protein-peptidecomplexes differentially expressed by the target cell relative to anormal cell, wherein the first binding substances are antibodies orantigen binding fragments thereof; b) isolating second bindingsubstances that specifically bind to the first binding substances,wherein the second binding substances are peptides or anti-idiotypicantibodies; and c) incorporating the second binding substances into theimmunogenic composition.
 2. The method of claim 1, wherein the isolatedfirst binding substances are a single species.
 3. The method of claim 1,wherein the isolated first binding substances are a mixture of species.4. The method of claim 1, wherein the second binding substances are asingle species.
 5. The method of claim 1, wherein the second bindingsubstances are a mixture of species.
 6. The method of claim 1, whereinthe isolated first binding substances are obtained by screening a phagedisplay library.
 7. The method of claim 1, wherein the isolated secondbinding substances are obtained by screening a phage display library. 8.The method of claim 1, wherein the antigen binding fragments are Fabs.9. The method of claim 1, wherein the antigen binding fragments aresingle chain Fvs.
 10. The method of claim 1, wherein the isolated secondbinding substances are peptides.
 11. The method of claim 10, wherein thepeptides are 30 amino acids or fewer in length.
 12. The method of claim10, wherein the peptides are from 4 to 20 amino acids in length.
 13. Themethod of claim 10, wherein the second binding substances are from 7 to15 amino acids in length.
 14. The method of claim 1, wherein the heatshock protein-peptide complexes comprise complexes of more than one heatshock protein.
 15. The method of claim 1, wherein the heat shockprotein-peptide complexes comprise a heat shock protein selected fromgp96, BiP, hsp70, hsc70, hsp60, hsp40 and hsp90.
 16. The method of claim1, wherein the heat shock protein is gp96.
 17. The method of claim 1,wherein the target cell is a tumor cell.
 18. The method of claim 17,wherein the tumor cell is from a sarcoma, a lymphoma, a leukemia, acarcinoma, or a melanoma.
 19. The method of claim 18, wherein thecarcinoma is selected from carcinoma of the breast, carcinoma of theprostate, ovarian carcinoma, carcinoma of the cervix, colon carcinoma,carcinoma of the lung, glioblastoma, and astrocytoma.
 20. The method ofclaim 1, wherein the target cell is an infected cell.
 21. The method ofclaim 20, wherein the infected cell contains a virus, a mycoplasma, aparasite, a protozoan, or a prion.
 22. The method of claim 1, wherein instep c) the second binding substances are further conjugated to acarrier.
 23. The method of claim 1, wherein in step c) the secondbinding substances are further complexed with a heat shock protein.